Not applicable.
The present invention relates to assays for sensitive and specific detection of analytes in biological, environmental, pharmaceutical, or industrial samples. Such assays have broad applicability for detection of infectious agents, including bacteria, viruses, fungi, parasites, and other organisms, and for analyzing normal or aberrant genes or gene expression. These assays are useful in fields including human and veterinary medicine, water and environmental quality, food safety, identification of the source of nucleic acids found in forensic samples, as well as paternity testing, and for improvement of plant and animal agricultural products.
Throughout this application, various patents, published patent applications and other publications are referenced and citations provided for them. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
A variety of methods are used in the art to detect and analyze analytes in biological samples. These methods include, among others, methods to identify and distinguish polynucleotide sequences, such as nucleic acid hybridization, methods to increase the quantity of polynucleotides, such as polymerase chain reaction or PCR (U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188), nucleic acid sequence based amplification or NASBA (U.S. Pat. Nos. 5,409,818; 5,130,238; and 5,554,517), transcription-mediated amplification or TMA (U.S. Pat. No. 5,437,990), self-sustained sequence replication or 3SR (Fahy, et al., PCR Methods and Appl. 1: 25-33, 1991), ligation chain reaction or LCR (e.g., U.S. Pat. Nos. 5,494,810 and 5,830,711), continuous amplification reaction or CAR (U.S. Pat. No. 6,027,897), linked linear amplification of nucleic acids or LLA (U.S. Pat. No. 6,027,923) and strand displacement amplification or SDA (U.S. Pat. Nos. 5,455,166; 5,712,124; 5,648,211; 5,631,147), and methods to increase a signal produced in the presence of a polynucleotide, such as rolling circle amplification or RCA (U.S. Pat. No. 5, 854,033), cycling probe reaction or CPR (e.g., U.S. Pat. Nos. 4,876,187 and 5,011,769 and 5,660,988), branched chain amplification (e.g., U.S. Pat. Nos. 4,775,619 and 5,118,605 and 5,380,833 and 5,629,153). Many of these methods are discussed by Brow, et al. in U.S. Pat. No. 6,001,567, which is incorporated herein by reference.
Much work has also been done on using Q-beta (Q-beta) replicase, an RNA-dependent RNA polymerase for increasing the quantity of a polynucleotide or amplifying a signal produced in its presence (e.g., see U.S. Pat. Nos. 4,786,600; 4,957,858; 5,112,734; 5,118,801; 5,312,728; 5,356,774; 5,364,760; 5,472,840; 5,503,979; 5,556,751; 5,556,769; 5,602,001; 5,616,459; 5,620,851; 5,620,870; 5,629,156; 5,631,129; 5,652,107; 5,686,243; 5,750,338; 5,759,773; 5,763,171; 5,763,186; 5,780,273; 5,800,994; 5,807,674; 5,837,466; 5,871,976; 5,959,095; 6,001,570; European Patent Nos. 0266399; 0346594; 0386228; 0436644; 0473693). Some researchers believe it is the most sensitive system known.
Methods using Q-beta replicase have been proposed for detecting of a wide variety of analytes, including nucleic acids (DNAs and RNAs) and segments of nucleic acids; proteins, including glycoproteins and lipoproteins, enzymes, hormones, receptors, antigens, and antibodies; and polysaccharides. For example, Chu, et al. (U.S. Pat. Nos. 4,957,858 and 5,364,760) disclosed methods in which a substrate for Q-beta replicase was attached by various methods to an affinity molecule for an analyte. Following binding of the affinity molecule to the analyte and washing to remove unbound affinity molecules, the substrate was released from the affinity molecule by various method and was replicated by Q-beta replicase. Thus, replication of the substrate served as signaling system for the presence of the analyte. Most of the other work with Q-beta replicase has been limited to detecting nucleic acid analytes.
Q-beta replicase is remarkable because, from a small number of template strands, it can initiate in vitro synthesis of a large number of product strands (Haruna, I., and Spiegelman, S., Proc. Nat. Acad. Sci. USA, 54: 579-587, 1965; Science, 150: 884-886, 1965). A 100,000-fold increase in RNA can occur during a ten-minute reaction (Kramer, F. R., et al., J. Mol. Biol., 89: 719-736, 1974). This striking amplification is the consequence of an autocatalytic reaction mechanism. Single-stranded RNAs serve as templates for the synthesis of complementary single-stranded products. Both the product strand and the template strand are released from the replication complex and are free to serve as templates in subsequent rounds of synthesis. Consequently, the number of RNA strands increases exponentially as the reaction proceeds. The autocatalytic reaction proceeds at an exponential rate until the number of autocatalytically replicatable RNA molecules exceeds the number of active enzyme molecules in the reactions. After that point, the amount of autocatalytically replicatable RNA increases linearly with time. As a consequence, in reactions given a sufficient period of time to reach this linear phase (for example 15 minutes for 100 molecules), the amount of amplified product RNA will be directly related to the logarithm of the number of autocatalytically replicatable RNAs initially added (Lizardi, et al., Nature Biotechnology, 6: 1197-1202, 1988). Since the initial number of autocatalytically replicatable RNA probes is proportional to the amount of target, the amount of target present in the sample being examined may be quantified over a very wide range.
In vitro, Q-beta replicase can utilize a number of other RNA molecules besides the Q-beta genome as templates. One such template, termed midivariant RNA (MDV), discovered as a naturally occurring product in Q-beta replicase reactions, has been used for making amplifiable reporter probes for nucleic acid hybridization assays (e.g., U.S. Pat. No. 4,786,600) . These reporter probes were made by inserting a target-specific probe sequence into an MDV molecule in a site such that it: 1) permits the MDV probe to specifically hybridize to its intended target nucleic acid, and 2) remains replicatable by Q-beta replicase in spite of the additional probe sequence. The MDV serves as an amplifiable detection ligand. One billion or more progeny molecules can be produced from a single starting template recombinant MDV molecule in approximately 30 minutes. Thus, a very large number of detection ligands (MDV RNA molecules) can be produced from very few hybridized reporter probes.
Theoretically, this permits the development of extremely sensitive nucleic acid hybridization assays; that is, assays which are capable of detecting the presence of very few target molecules (or organisms) in a test sample. However, assay sensitivity is a function not only of the amount of signal that can be generated for a given amount of target nucleic acid, but also of the amount of xe2x80x9cbackgroundxe2x80x9d signal that is generated even in the absence of target nucleic acid. The presence of background limits the sensitivity of assays at low target concentrations. Target induced signal must be significantly greater than background in order for assays to be considered reliable. Background has been a serious problem for assays using Q-beta replicase, in part because even a single replicatable RNA molecule will be replicated by the enzyme at an exponential rate.
For example, although Lizardi et al. (Nature Biotechnology, 6: 1197-1202, 1988) showed that MDV-1 RNA with a sequence for a protozoan parasite embedded within it was capable of exponential amplification by Q-beta replicase, they concluded that xe2x80x9cpractical assays employing recombinant RNAs did not exist yetxe2x80x9d using this method because nonspecifically bound probes served as templates for amplification. According to the methods and format used, the recombinant RNA probes bound to the hybridization surface in sufficient quantities to be amplified by Q-beta replicase even in the absence of the protozoan target. They suggested that other methods were necessary for lowering the level of xe2x80x9cbackgroundxe2x80x9d signal due to nonspecifically bound recombinant RNA probes.
In addition to the problem of background due to nonspecific binding, researchers using heterologous sequences embedded within replicable Q-beta substrates like MDV also encountered another problem: the probe sequence is viewed as foreign by the enzyme and affects the ability of the RNA to be efficiently replicated, or is spontaneously deleted during replication. Deletion events affect the rate of replication and occur randomly with time. When deletion events occur, the level of the RNA products obtained in the linear phase of the amplification cannot be used to assess target level. Also, a further type of background, common with autocatalytically replicatable amplification systems, is xe2x80x9cunprimedxe2x80x9d activity of the enzyme itself (i.e., the enzyme makes new templates from mononucleotides de novo).
Q-beta replicase is composed of one phage-encoded subunit and three host-encoded subunits (Blumenthal, T. and Carmichael, G. G., Ann. Rev. Biochem., 48: 525-548, 1979). One source of background originates with the enzyme itself. For example, Q-beta replicase synthesizes new RNA species in the absence of extraneously added substrate (Biebricher, C. K., and Luce, R., Biochemistry, 32:4848-4854, 1993). No matter how poorly the new RNA replicates, once formed, it is amplified and optimized, although in an irreproducible manner. The products differ from experiment to experiment even when conditions are identical. This xe2x80x9cevolutionxe2x80x9d of RNA molecules toward those which are more optimized for replication occurs by random addition of nucleotides to the ends of the new RNAs, by mutation, which can occur at an elevated rate, and by recombination of RNA molecules (Munishkin, A.V., et al., J. Mol. Biol., 221: 463-472, 1991; Biebricher, C. K., and Luce, R., EMBO Journal, 11: 5129-5135, 1992).
Another significant source of background signal in nucleic acid probe systems using Q-beta replicase has been the presence in some Q-beta replicase preparations of contaminating RNA termed xe2x80x9cwild-typexe2x80x9d or xe2x80x9cendogenousxe2x80x9d variant RNAs, which probably originated as just discussed, and which require use of special purification protocols to remove (e.g., see DiFrancesco, U.S. Pat. No. Re.35,443).
U.S. Pat. No. 5,750,338, attempted to reduce background due to nonspecific binding of the probe to solid supports using a method by which the target-probe complex was reversibly bound to the support (xe2x80x9creversible target capturexe2x80x9d). After hybridization and immobilization, the complex is eluted from the support, which is then discarded with the nonspecifically bound probe. The target-probe is then recaptured on fresh support. This process may be repeated several times to produce a significant reduction in the amount of non-hybridized probe. The method is difficult and tedious, and usually did not reduce background sufficiently to achieve the sensitivity that is desired from such assays.
In U.S. Pat. Nos. 5,364,760 and 4,957,858, Chu et al. disclosed other methods to try to reduce background and develop sensitive analyte-specific assays using Q-beta replicase. For example, Chu et al. disclosed the use of xe2x80x9csmartxe2x80x9d probes. These probes were called xe2x80x9csmartxe2x80x9d because, ideally, the replicative RNA of a hybrid is replicated by the RNA-dependent RNA polymerase if and only if the affinity molecule portion hybridized to its analyte and the replicative RNA portion was released. That this was not completely successful is indicated by the inventors"" statement that this occurs xe2x80x9cbut for unavoidable xe2x80x9cnon-specific bindingxe2x80x9d of affinity molecule or replicative RNA (via the RNA portion or first linking moiety portion thereof), which give rise to xe2x80x9cbackgroundxe2x80x9d.xe2x80x9d In addition, the synthesis of many of the probes that they used required several steps, increasing the cost and labor to produce them, and many contained disulfide linkages, which can be cleaved prematurely by reducing agents, such as glutathione, which occur naturally in biological samples, and which could lead to nonspecific signals.
In U.S. Pat. No. 5,763,171, Stefano discloses a method for detecting the presence of target nucleic acid in a sample using probes comprising a first section that is replicatable in the presence of an RNA-directed RNA polymerase, such as Q-beta replicase, and a second section that makes the first section incapable of replication. Ideally, the presence of a target sequence in a sample, in the presence of a release agent, results in target-specific release of the first section in a form that can be replicated by the RNA-directed RNA polymerase. Then, the released first section, which can be, for example MDV-1 RNA, is replicated under suitable reaction conditions and detected by some method known in the art. According to Stefano, the release method may take many forms, such as, by way of example, the enzyme RNase H or a ribozyme. Ideally, the first section of the probe is separated from or released from the second section only in the presence of the target sequence.
The state of the art at the time of filing of Stefano""s xe2x80x9c171xe2x80x9d patent (see U.S. Pat. No. 5,763,171 and references therein), taught that linking a target-complementary sequence to either the 3xe2x80x2-end or the 5xe2x80x2-end of an autocatalytically replicatable RNA, such as MDV-1, via the phosphodiester linkage normally found in RNAs xe2x80x9chas been reported to strongly inhibit replicationxe2x80x9d by an RNA-directed RNA polymerase like Q-beta replicase. Since then, additional work in the art has demonstrated that replicatable RNAs having a polynucleotide linked via a phosphodiester bond to its 5xe2x80x2-end can still be substrates for replication by Q-beta replicase. Also, although attaching a polynucleotide via a phosphodiester bond to the 3xe2x80x2-end of a replicatable RNA may inhibit replication, this inhibition is somehow quickly overcome. It is believed in the art that this occurs by replication of the first strand, and then either, subsequent occasional premature termination during replication of the second strand (i.e., the Q-beta replicase enzyme occasionally xe2x80x9cfalls offxe2x80x9d of the template before synthesis of a complete RNA strand), or by occasional random breakage of template or product RNA. In either case, a substrate for replication is generated. Also, the rate of regeneration of replicatable RNA substrates may be increased by another characteristic of Q-beta replicase: if the normal end sequences of the substrate are not present, as might occur by premature termination or breakage of RNA, one enzymatic activity of Q-beta replicase is the random addition of nucleotides to the ends of the RNA. If any of these enzymatic activities results in even a single molecule of a xe2x80x9ccorrectxe2x80x9d or nearly correct (i.e., optimal) substrate, this substrate will be rapidly replicated and lead to a nonspecific signal.
It appears that Stefano also changed his ideas about the possibility of using a probe having a target-complementary sequence linked to either the 3xe2x80x2-end or the 5xe2x80x2-end of an autocatalytically replicatable RNA in the ways he envisioned in the xe2x80x9c171xe2x80x9d patent. In U.S. Pat. No.5,556,751, which is a continuation-in-part of a patent filed later than the original patent application on which U.S. Pat. No. 5,763,171 is based, Stefano explicitly states that xe2x80x9cif the probe sequence is appended to either end of the MDV sequence, then it is not replicated along with the MDV sequence.xe2x80x9d Thus, in U.S. Pat. No. 5,556,751, Stefano proposed assays in which a target-complementary probe sequence was appended to the 3xe2x80x2-terminus of a propidium iodide-resistant MDV sequence because he by then realized and disclosed that the target-complementary sequence on the 3xe2x80x2-terminus is lost when the MDV-probe sequence is replicated by Q-beta replicase, even without using any kind of release method. He further states in the xe2x80x9c751 xe2x80x9d patent: xe2x80x9cThe advantage of designing the MDV probes in this fashion is that the probe sequence does not also need to be resistant to the inhibitory agent, since it is not replicated by Q-beta replicase.xe2x80x9d Thus, the increased specificity and reduced background were not obtained as intended by the invention disclosed in the xe2x80x9c171xe2x80x9d patent.
Some of the problems that result in background and low signal to noise ratios using RNA-directed RNA polymerases have been addressed by using split probes. For example, U.S. Pat. No. 5,631,129 uses Q-beta replicase to extend an RNA primer through a short target region of about 20-500 nucleotides. Two RNA primers are prepared. The first primer contains the first 157 nucleotides (at the 5xe2x80x2-end) of the minus-strand of Q-beta MDV-1 RNA followed by 10-50 nucleotides that are complementary to the target RNA over a region extending 5xe2x80x2 from the 3xe2x80x2 end of the site of the target sequence. The second primer contains the first 61 nucleotides (at the 5xe2x80x2 end) of the plus-strand of Q-beta MDV-1 RNA followed by 10-50 nucleotides that are identical to the target RNA over a region extending 3xe2x80x2 from the 5xe2x80x2-end of the target sequence. The two primers are hybridized to a target sequence, if present in a sample, and primer extension occurs in the presence of Q-beta replicase. Then, the second primer-extension product is released from the template by heating to 70xc2x0 C. for 1 minute and quick-cooling on ice. Another aliquot of Q-beta replicase is added and amplification of the target RNA then proceeds autocatalytically by incubating at 37xc2x0 C. for 20 min. The resulting mixture can then be assayed for the production of MDV-1 RNA that contains an insert that corresponds to the desired target sequence. Thus, the method uses portions of autocatalytically replicatable RNAs and results in the production of recombinant replicatable RNAs, permitting amplification of a target nucleic acid of interest.
Although the primer extension method of U.S. Pat. No. 5,631,129 should reduce background signal, the activity of Q-beta replicase in primer extension appears to be very low. Since double-stranded RNA is not a template for replication by Q-beta replicase, the 70xc2x0 C. denaturation step may be required for replication. The method also appears to have some of the same disadvantages as PCR, which is influenced by many variables. For example, target DNA length and secondary structure, primer length, specificity and design, primer and dNTP concentrations, and buffer composition can all affect PCR and would be expected to influence primer extension prior to replication by Q-beta replicase.
In U.S. Pat. No. 5,759,773, Tyagi, et al. described still another approach. Tyagi et al. designed xe2x80x9cbinary probesxe2x80x9d which consisted of two separate molecules, neither of which could be amplified by itself because neither contained all of the elements of sequence and structure that were required for replication by Q-beta replicase. Each probe contained half of an MDV substrate and part of a target-complementary sequence. The target-complementary sequences of the two probes were contiguous when hybridized to the target. Thus, ligation of the two binary probes when they were hybridized to the target resulted in a complete recombinant MDV that was capable of replication in the presence of Q-beta replicase. Since non-hybridized probes that were not aligned on a target had a very low probability of being ligated in solution, the background was reduced. The inventors also used other steps, such as cleavable capture probes to release hybrids from surfaces, to reduce background still further. Somewhat similarly, U.S. Pat. No. 5,959,095 utilized DNA binary MDV probes which contained contiguous target-complementary sequences, and one of which served to immobilize the target on the surface. The ligated recombinant MDV DNA was transcribed into RNA, using a phage promoter sequence on one of the binary MDV probes before replication with replicase.
The binary probe method of Tyagi et al. (U.S. Pat. No. 5,759,773) appears to have promise. The only apparent problems are the amount of labor involved to perform the number of steps involved and the potential for background due to nonspecific ligation; since the thermostable ligases which are currently used for LCR are not active on RNA, additional work is required before assays with RNA probes can be carried out at higher temperatures and higher stringency. The lability of RNA at elevated temperature increases the difficulty of developing such assays, and argues in favor of binary DNA probes, as described in U.S. Pat. No. 5,959,095. However, based on past experience, it is likely that the methods of Tyagi et al. will have lower backgrounds than methods involving ligation of probes hybridized to a surface.
In European Patent No. EP 051 9053B1, Dimond et al. also disclosed some methods using split or binary probes which are similar to those described in U.S. Pat. Nos. 5,631,129 and 5,759,773 discussed above, except that Dimond et al. used xe2x80x9ccomplex templatesxe2x80x9d which comprise at least one 2xe2x80x2-deoxyribonucleotide instead of RNA templates. Their disclosures were based on the fact that Q-beta replicase also has DNA-dependent RNA polymerase activity in the presence of greater than 0.5 mM concentration of manganese, cobalt or zinc divalent cations. In addition to disadvantages of the methods of U.S. Pat. Nos. 5,631,129 and 5,759,773, the assays of Dimond et al. are likely to also suffer from decreased reaction rates and lower specificity and accuracy compared to assays using Q-beta replicase in the presence of magnesium divalent cations. For example, as reviewed by Blumenthal and Carmichael (Ann. Rev. Biochem., 48: 525-548, 1979), adding manganese to the reaction mixture is one of the ways to overcome the template specificity of Q-beta replicase.
The above background, including references cited herein, summarize the state of the art pertaining to the use of RNA-directed RNA polymerases such as Q-beta replicase as of the date of the present invention. This summary shows that RNA-directed RNA polymerases such as Q-beta replicase are remarkable in being able to exponentially amplify even a single molecule by a billion-fold or more in about 30 minutes. In the absence of nonspecific background signals, these assays would permit quantification of target polynucleotide sequences over a broad concentration range of target in a sample. However, the art also shows that the properties of RNA-directed RNA polymerases result in special problems that still must be dealt with and overcome in order to develop sensitive, but specific and meaningful assays to detect target nucleic acid sequences in a sample. Among the properties causing special difficulties are the following: (1) since even a single substrate molecule is amplified, steps must be designed to eliminate background due to nonspecific binding of substrate molecules to solid surfaces or other molecules in the assay reaction mixture and prevent carryover contamination to subsequent assays; and (2) since target-complementary polynucleotide sequences or other eheterologous polynucleotide sequences that are inserted within a substrate for Q-beta replicase can be deleted during the replication reaction, and the amplification signal obtained need not be specific for the target, strategies must be implemented to ensure that the assay results are target-specific. In brief, the only assay methods which appear to have been successful using Q-beta replicase are those which use some form of a binary probe, such that different parts of a polynucleotide that are required for replication are joined together only in the presence of a target. Such assays include, for example, those disclosed in U.S. Pat. No. 5,631,129, U.S. Pat. No. 5,759,773 and No. 5,959,095, and some of the embodiments in European Patent No. EP0519053B1. However, as discussed above, even these latter assays require additional work to be accepted for routine use by those who work in the various fields of use of the present invention.
What is needed in the art are assays with the sensitivity of exponential amplification obtained with RNA-dependent RNA polymerases like Q-beta replicase, yet which are easier to control and design, and which yield lower backgrounds than assays which amplify both target and replicase substrate sequences.
What is needed in the art is a method that uses a replicase to generate a signal in a target-specific manner, but which does not amplify the target.
What is needed is a method for using a replicase which permits accurate quantification of the amount of an analyte in a sample.
What is needed is a method for using a replicase that permits multiplex assays for multiple analytes in a sample, including multiplex assays for accurate quantification of different analytes.
What is needed is a method for using a replicase that permits assays for a broad variety of analytes.
What is needed is a method that can be used in homogeneous assays for an analyte in a sample.
The above background, including references cited herein, being a general description of the state of the art pertaining to the present invention, we now summarize the invention below.
In this description and in the appended claims, I sometimes refer to a molecule in the singular (e.g., xe2x80x9ca reporter probexe2x80x9d). The same applies to a complex (e.g., xe2x80x9ca reporter probe-target hybridxe2x80x9d). It will be understood that many copies are in fact used or made. I sometimes utilize the plural rather than stating xe2x80x9ccopies of,xe2x80x9d which is cumbersome. Persons skilled in the art will understand the meaning from the context.
The assays of the present invention are signal amplification assays for detecting the presence of at least one analyte or, preferably, multiple analytes in a sample. The assays of the invention are also for quantifying analyte molecules in a sample, or for identifying analytes in a sample. These assays include at least one improvement, but preferably, a combination of improvements, to reduce background and improve sensitivity and specificity.
The present invention comprises compositions and methods for assaying for an analyte in a sample, said methods comprising the steps of:
(a) incubating a sample with a reporter probe under conditions and for a time so as to permit binding of said reporter probe with analyte, if present in sample, said reporter probe comprising a first portion, wherein said first portion is a polynucleotide that encodes at least part of a sequence for a replicase substrate, and a second portion, wherein said second portion has affinity for the analyte under binding conditions, and wherein said reporter probe is not a substrate for replication by said replicase; and
(b) incubating said reporter probe which binds to analyte with a composition having nuclease activity, wherein said composition releases all parts of said first portion of said reporter probe from said second portion of said reporter probe; and
(c) incubating said released parts of said first portion of said reporter probe with a composition having ligase activity so as to form a polynucleotide which encodes a complete substrate for replication by a replicase, and providing conditions so as to generate said substrate; and
(d) incubating said substrate with a replicase under replication conditions; and
(e) detecting the products of said replication.
Preferred embodiments of the invention use a reporter probe comprising monoribonucleosides.
Embodiments of assays which use a reporter probe having a first portion comprising monodeoxyribonucleosides additionally comprise an in vitro transcription step in order to generate an RNA substrate for replication by a replicase.
Preferred embodiments of the invention are homogenous assays. In other preferred embodiments, unbound reporter probe molecules are separated from reporter probe molecules bound to analyte, if analyte is present in the sample, prior to contacting reporter probe molecules which are bound to analyte with a nuclease. Preferably, the separation is accomplished using at least one capture probe which binds to analyte and which can be immobilized on a surface.
The invention is not limited by the nuclease used to release the parts of the first portion of the reporter probe from the second portion of the reporter probe. Preferred embodiments use ribonuclease H (RNase H) or a 5xe2x80x2 nuclease as the composition having nuclease activity, but other compositions can also be used.
The invention also includes assays for quantification of analytes, including, but not limited to, nucleic acid or polynucleotide analytes, over a broad concentration range of the analyte in a sample.
The invention further includes assays for simultaneous detection of the presence of any of multiple different target polynucleotide sequences in a sample.
The inventor believes, that in addition to solving the types of problems encountered using Q-beta replicase, the present invention also provides at least one advantage, and often multiple advantages, over other analyte-specific assays of the prior art. Among the advantages of the present invention for assays for nucleic acid analytes are the following:
1. The methods of the present invention do not involve nucleic acid synthesis using the target nucleic acid as a template. For methods involving DNA-dependent and/or RNA-dependent polymerization of a target sequence (e.g., PCR, NASBA, TMA, 3SR), the priming efficiency, priming specificity, target length, and processivity of polymerization can differ for different templates based on sequence and structure. Therefore, the primers and assay conditions must be carefully optimized for each target, and even so, the results vary for each template. These variables cause special difficulties in trying to quantify nucleic acid targets, especially for multiplex assays for multiple targets. Even for other assays using replicatable recombinant RNA probes in which different target-complementary sequences are inserted in a Q-beta replicase substrate like MDV-1 RNA, the replication efficiency of each probe usually differs, leading to differences in quantitative results.
Further, unless special methods and sample handling procedures are implemented in the user""s laboratory, assays that comprise synthesizing a target nucleic acid sequence can result in spread of the amplified product to negative samples, which is referred to in the art as xe2x80x9ccarryover contamination.xe2x80x9d For example, it is well known that carryover contamination can be a serious problem for diagnostic laboratories that perform PCR assays routinely, and a variety of compositions and methods are available to try to avoid problems of PCR carryover contamination.
In contrast, the methods of the present invention are based on the well-understood principles of hybridization of complementary sequences and nuclease digestion using enzymes, such as ribonuclease H, which do not produce significantly different results for different target sequences. Since the RNA substrate encoded by the first portion of the reporter probe can be identical whatever target-complementary sequence is used in the second portion, quantification of different analytes is directly comparable.
Also, since the target nucleic acid is not synthesized in assays of the present invention and, in fact, the target-complementary sequence of the second portion is destroyed by nuclease digestion in most embodiments of the invention, there is no chance for carryover contamination of amplified target nucleic acid to other samples.
2. The assays of the present invention do not require thermocycling like assays such as PCR and LCR. The need for a thermocycler increases expense and availability of assays and limits the number of assays that can be performed to the capacity of the thermocycler.
In addition, thermocycling introduces numerous variables into the assays, which can have dramatic effects on specificity and sensitivity of assays. For example, each brand of thermocycler, and sometimes even each individual machine, can have different ramp times (i.e., different rates of change of temperature) and differences in temperature in different parts of the heating block, which can also change over time. Different thermostable enzymes, and even different preparations of the same enzyme, can have different activities and different half lives at any given temperature, and can be affected differently (e.g., inactivated by different amounts) by the temperature cycling. A relatively small difference in the relative enzyme activity or in the rate of enzyme inactivation during one cycle results in a large difference in amplification yield after multiple cycles. For example, in a PCR reaction with 30 amplification cycles and a 10% difference in yield per cycle, the difference in final yield would be more than 20-fold after 30 cycles. Depending on the amount of analyte in the sample and the sensitivity of the detection method used, this could be the difference between a positive assay and a false negative.
In contrast, the methods of the present invention do not use thermocycling. Since a thermocycler isn""t needed, assays of the present invention are less expensive, and the number of assays that can be performed is not limited by availability or capacity of the thermocycler.
Also, most embodiments of the present invention do not use steps in which an enzyme component is exposed to an elevated temperature, minimizing the chance for enzyme inactivation or variability of assay results. All enzymatic steps of the present invention can be performed under isothermal conditions. In some embodiments, the assays can even be designed to be performed under xe2x80x9cambientxe2x80x9d conditions (i.e., in the field). In those embodiments in which a higher temperature is used for certain steps in order to achieve a higher stringency, the steps are still carried out under isothermal conditions, so consistent results are obtained, and sample-to-sample quantification comparisons can be made.
3. The assays of the present invention produce low background signals and result in exponential amplification. The cycling probe reaction (CPR) generates a signal by ribonuclease H cleavage of each probe that hybridizes to the nucleic acid analyte, if analyte is present in the sample. Once a probe is cleaved, the resulting fragments are too short to remain hybridized to the analyte, permitting another probe to anneal and be cleaved, thereby increasing the signal. The maximum signal obtain is only about 1000-fold to 10,000-fold over background. The CAR assay, which relies on transcription, also does not result in signal amplification as high as can be obtained by PCR or NASBA.
In contrast, embodiments of the assays of the present invention use an exponential replication mechanism, resulting in highly sensitive assays because of production of large amounts of amplified RNA and a low background signals in the absence of a specific target analyte.
4. The assays of the present invention can be designed to detect analytes that are not nucleic acids. Most of the other assays discussed above use one or more steps that limits their ability to be used to detect analytes other than nucleic acids. The two exceptions are Q-beta replicase assays (U.S. Pat. Nos. 4,957,858 and 5,364,760) and some embodiments of Rolling Circle Amplification (U.S. Pat. No. 5,854,033).
In contrast to other assays, the present invention discloses how proven methods for making and using nucleic acids as analyte-binding substances can be used in assays to detect non-nucleic acid analytes. Thus, reporter probes and methods used for embodiments of the present invention for nucleic acid analytes can also be used to make sensitive and specific assays for other analytes of almost any composition. Also, the invention is not limited to reporter probes comprising nucleic acids for detection of non-nucleic acid analytes. By using a linking oligonucleotide to join the analyte-binding substance of the second portion of the reporter probe to the signal-producing substrate encoded by the first portion, a variety of non-nucleic acid analyte-binding substances can be used to make sensitive and specific assays for a variety of analytes.
It is an object of this invention to provide sensitive and specific assays for a variety of analytes.
It is a further object of the invention to provide assays that permit detection of any of multiple different analytes in the same sample.
It is a further object of the invention to provide assays that permit quantification of analytes over a broad range of analyte concentrations.
It is a further object of the invention to provide homogenous assays.
It is a further object to provide compositions and kits for performing the methods of the invention.